1. Field of the Invention
The invention relates to methods for determining or predicting response to cancer therapy in an individual. In particular, the invention relates to methods for using image analysis to assess the efficacy of chemotherapeutic and chemopreventative agents in a human in need of treatment with such agents by detecting expression levels of biological makers associated with senescence, apoptosis or terminal differentiation. More specifically, the invention provides methods where the amount of the senescence, apoptosis or terminal differentiation marker is quantitated in tissue and cell samples removed from an individual before and after exposure to the chemotherapeutic or chemopreventative agent.
2. Background of the Invention
A primary goal of cancer therapy is to selectively kill or inhibit the uncontrolled growth of malignant cells while not adversely affecting normal cells. Traditional chemotherapeutic agents are highly cytotoxic agents that preferably have greater affinity for malignant cells than for normal cells, or at least preferentially affect malignant cells based on their high rate of metabolic activity. However, these agents often harm normal cells.
In the use of anticancer drugs, monoclonal antibodies, or chemopreventive agents, growth arrest, terminal differentiation and cell death of the cancerous or precancerous cells is intended (Mendelsohn, 1990, Semin. Cancer Biol. 1:339-44; Hancock et al., 1991, Cancer Res. 51:4575-80; Arteaga et al., 1994, Cancer Res., 54:3758-65; Pietras et al., 1994, Oncogene 9:1829-38; Bacus et al., 1997, Anal. Quant. Cytol. Histol. 19:316-28; Bacus et al., 1999, Breast J.; Baselga et al., 1999, Proceedings of AACR NCI EORTC International Conference, Abstract 98; Cobleigh et al., 1999, J. Clin. Oncol. 17:2639-48; DiGiovanna, 1999, PPO Updates: Princ. Practice Oncol. 13:1-9; Hortobagyi, 1999, J. Clin. Oncol. 17:25-29; Shak, 1999, Semin. Oncol. 26:71-77; Sliwkowski et al., 1999, Semin. Oncol. 26:60-70; Vincent et al., 2000, Cancer Chemother. Pharmacol. 45: 231-38). Drug-induced growth arrest or cell death is often characterized by morphological and biochemical changes associated with programmed cell death or terminal differentiation (as opposed to mitotic cell death).
Although chemotherapeutic drugs can be administered at doses high enough to bring about cell death, such doses typically produce deleterious effects on normal as well as tumor cells. Differentiating agents, and lower doses of chemotherapeutic drugs and agents frequently results in growth arrest rather than cell death; such arrest can be followed by apoptosis and sell death, or continued proliferation once the chemotherapeutic drugs are withdrawn. Administration of cytotoxic and chemotherapeutic drugs or ionizing radiation may also induce transient growth arrest, a state which depends largely on the function of p53 and a p53-regulated cyclin-dependent kinase inhibitors (such as p16, p27, and p19) or growth inhibitors (such as TGF-β, IL-4, and IL-6). Upon removal of the chemotherapeutic drug, cells subjected to the drug treatment will eventually resume division and either continue to proliferate or die. Some drug-treated tumor cells undergo prolonged growth arrest and fail to resume cell division upon release from the drug.
In normal cells, terminal proliferation arrest may result from terminal differentiation or replicative senescence. Senescence is a physiological process that limits the proliferative span of normal cells. A commonly-used marker of senescence in human cells is expression of senescence-associated β-galactosidase (SA-β-Gal). This protein has been shown to correlate with senescence in aging cell cultures in vitro and with cells in vivo. Terminal proliferation arrest in normal cells can be rapidly induced by treatment with DNA-damaging drugs or γ-irradiation and is accompanied by the morphological features of senescence and the induction of SA-β-Gal. Accelerated senescence is likely to be a protective response of cells to carcinogenic impact. Similar to other damage responses of normal cells—such as quiescence and apoptosis—senescence-like terminal proliferation arrest involves the function of proteins such as wild-type p53 and the up-regulation of such proteins as p21WAF1, p16, p19, and p27 (Kwok and Sutherland, 1989, J. Natl. Cancer Inst. 81:1020-24; Kwok and Sutherland, 1991, Int. J. Cancer 49:73-76; Kastan et al., 1991, Cancer Res. 51:6304-11; Lane, 1992, Nature 358:15-16; Kuerbitz et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7491-95; Gu et al., 1993, Nature 366:707-10; Halevy et al., 1995, Science 267:1018-21; Sherr and Roberts, 1995, Genes Dev. 9:1149-63; Luo et al., 1995, Nature 375:159-61; Dimri et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:9363-67; Bacus et al., 1996, Oncogene 12:2535-47; Liu et al., 1996, Cancer Res. 56:31-35; Wang et al., 1998, Oncogene 17:1923-30; Chang et al., 1999, Oncogene 18:4808-18; Hong et al., 1999, Cancer Res. 59:2847-52; Sionov and Haupt, 1999, Oncogene 18:6145-57; Wouters et al., 1999, Oncogene 18:6540-45).
Tumor cells appear to have retained the ability to enter senescence and terminal proliferation arrest. Treatment of tumor cells with different classes of agents that affect cell differentiation, and anticancer agents, readily induces morphological, enzymatic, and other changes characteristic of senescence (such as the up-regulation of p53, p21, p27, p16, TGF-β, IL-4, IL-6, and SA-β-Gal). This senescence-like phenotype (SLP) distinguishes cells that will become stably growth-arrested from cells that will recover from drug exposure and continue to proliferate. Thus, the induction of senescence-like terminal proliferation arrest provides an important determinant of treatment response in tumor cells.
Apoptosis is generally regarded as an active suicidal response to various physiological or pathological stimuli. Recent studies have shown that a variety of DNA-damaging agents, including X-ray irradiation and several chemotherapeutic drugs (e.g., alkylating agents and topoisomerase II inhibitors) cause necrosis or initiate pathways leading to apoptosis. The exact mechanism by which apoptosis is induced by these agents is not yet known. However, expression of the suppressor gene p53 has been implicated in this process (Kwok and Sutherland, 1989, J. Natl. Cancer Inst. 81:1020-24; Kwok and Sutherland, 1991, Int. J. Cancer 49:73-76; Lane, 1992, Nature 358:15-16; Kuerbitz et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7491-95; Luo et al., 1995, Nature 375:159-61; Liu et al., 1996, Cancer Res. 56:31-35; Mellinghoff and Sawyers, 2000, PPO Updates 14:1-11). In addition, the up-regulation of caspases (e.g., caspase 9 or caspase 3) or their chaperone molecules (e.g., heat shock protein 60) has been associated with apoptosis.
In cells undergoing apoptosis, DNA-damaging stimuli can result in an elevation of intracellular p53 protein levels. Increased levels of wild-type p53, in turn, inhibit the cell cycle at G1, thus permitting the damaged cell to undergo DNA repair. However, if the damaged cell is unable to undergo DNA repair, p53 can trigger programmed cell death. It is this ability to trigger programmed cell death that contributes to the induction of tumor cell death following exposure to chemotherapeutic agents.
Increased levels of p53 can also lead to the activation of a number of genes that contain wild-type p53-binding sequences, including the MDM-2 oncogene, Bax, and the WAF1/CIP1 gene. The WAF1/CIP1 gene encodes a protein having a Mr of 21,000 that associates with cyclin-Cdk complexes and is capable of inhibiting kinase activity associated with these complexes. A major target of WAF (p21 or CIP) inhibition is the cyclin E-Cdk2 kinase complex whose activity is required for G0 to S phase cell cycle progression. The WAF1/CIP1 gene is transcriptionally activated in response to DNA-damaging agents that trigger G1 arrest or apoptosis in cells with wild-type p53 but not in tumor cells harboring deletions or mutations of the p53 gene. However, WAF1/CIP1 has also been shown to be up-regulated in cells undergoing differentiation or cell cycle arrest by a p53-independent mechanism.
Thus, there are a variety of cellular markers of senescence, apoptosis and terminal proliferative arrest that can be used to detect the effects of chemotherapeutic and chemopreventative drugs and agents. These markers can be used to assess the success or failure of any particular chemotherapeutic or chemopreventative drug or agent, or combination thereof, to affect an anticancer effect on tumor cells in vivo.
In contrast to traditional anticancer methods, where chemotherapeutic drug treatment is undertaken as an adjunct to and after surgical intervention, neoadjuvant (or primary) chemotherapy consists of administering drugs as an initial treatment in cancer patients. One advantage of such an approach is that, in primary tumors of more than 3 cm this approach permits the use of conservative surgical procedures (as opposed to, e.g., mastectomy in breast cancer patients) in the majority of patients. Another advantage is that for many cancers, a partial and/or complete response is achieved in about two-thirds of all cases. Finally, since the majority of patients are responsive after two to three cycles of chemotherapeutic treatment, it is possible to monitor the in vivo efficacy of the chemotherapeutic regimen employed, which is important for a timely identification of those cancers which are non-responsive to chemotherapeutic treatment. Timely identification of non-responsive tumors, in turn, allows the clinician to limit the cancer patient's exposure to unnecessary side-effects and institute alternative treatments.
The efficacy of chemotherapeutic agents in treating particular cancers is unpredictable. In view of this unpredictability, it has not been possible to determine, prior to starting therapy, whether one or more selected agents would be active as anti-tumor agents or to render an accurate prognosis of course of treatment in an individual patient. It would be very desirable to be able to determine the efficacy of a proposed therapeutic agent (or combination of agents) in an individual patient. There is a need in the art for a method of assessing the efficacy of chemotherapeutic programs that is both time- and cost-effective and minimally traumatic to cancer patients.